Project Summary Isolation of Viable Human Sperm from Failed Microsurgical Testicular Biopsies Infertility is common; it affects 15% of the general population with 7.3 million American couples undergoing evaluation for infertility each year. Both women and men can be infertile. The most severe case of male infertility is where no sperm can be found in the ejaculate (10-20% of cases); a condition known as azoospermia. Sperm is produced in the testes. It is believed that the majority of men who do not have sperm in their ejaculate may have a minuscule number of sperm still being produced in their testicles. We have developed a novel methodology to detect and isolate a low number of sperm in the ejaculate. In this proposal, we will test this application to determine if usable sperm can be found where microsurgery, the current available treatment method, has failed. We will first compare the enzymatic processing of testis tissue with the mechanical shredding of a testis biopsy, to test which of these tissue preparation methods will result in a better separation of sperm from somatic and germ cells and thus yield a higher number of usable sperm. We will then test if removing the somatic cells from the testis biopsy, before sorting the specimen, will increase the number of sperm retrieved from men with severe infertility. Subsequently, we will also test if the sorted sperm, using the selected combination of fluorescent dyes, has any negative effect on sperm viability, motility, DNA structure, and mitochondrial function, as compared to unsorted sperm. Finally, we will compare the fertilization potential, embryo development, and pregnancy rates using sorted and unsorted sperm in an animal model, to ensure that the florescent dyes used, do not have any detrimental reproductive effects. These proposed experiments will provide the necessary information to start a clinical trial in humans, in which we can isolate live sperm for in vitro fertilization and utilize the downstream analysis for men where no sperm is found using the current available treatment methods.